Presomitic
mesoderm cultureThe experiments were
performed on the presomitic mesoderm of the 14- stage chick
embryo. The embryos were rinsed in the
Ca+2/Mg+2free
Tyrode's solution (8.0 g NaCl, 0.3 g KCl, 0.05 g
NaH2PO4*H2O
in 1L of dH2O).
The segment containing presomitic mesoderm was excised and
incubated in 0.25% tripsin-0.02% EDTA for 5 minutes. The
resultant mass of cells will be flushed through a narrow
bore pipet to remove ectoderm, endoderm, notocord, and
neural tube for at least 7 minutes. The digestion reaction
was terminated with 2 ml of calf serum and spun at 1750 rpm
for 7 minutes.

Treatment with FgF2 (Basic
Fibriblast Growth Factor)Twelve tissue culture
dishes were coated with 1% gelatin (Sigma) in deionized
water at 4oC overnight and gelatin was decanted
due to melting leaving an invisible layer of gelatin. The
cells were counted in 1:10 dilution using 10ml of Tripen
Blue. Cells were plated at density of 2.5 *
104in
20 ml. Three different concentrations of FgF2 were
tested:0.25 ml/10 ml, 1
ml/10ml, and 4 ml/10ml. Control was free of FgF2 and three
wells per each condition were used. Two hours after plating,
cells were flooded with 1.5 ml of the Dulbecco's Modified
Eagle's medium (DMEM) with 10% fetal calf serum and
antibiotics containing appropriate amount of FgF2.